Adult nervous systems consist of complex neuronal networks. The initiation of nerve fiber formation is one of the first crucial events in an elaborate process eventually resulting in the establishment of these highly organized structures. The regulation of nerve fiber growth is under the control of extracellular molecules including growth factors, cell adhesion molecules and extracellular matrix components. In addition a number of proteins have been implicated in the intracellular mechanisms that regulate neurite outgrowth such as the cytoskeletal protein actin (Tetzlaff and Bisby, 1990, Rest. Neurol. Neurosci. 1:189-196; Lewis and Bridgman, 1992, J. Cell Biol. 119:1219-1227), juvenile forms of tubulin (Miller et al., 1989, J. Neurosci. 9:1452-1463; Easter et al., 1993, J. Neurosci. 13:285-299), microtubule associated proteins (Lewis et al., 1989, Nature 342:498-505; Caceras et al., 1992, Neuron. 9:607-618), the developmentally regulated dendritic protein Drebrin (Shirao et al., 1992, Neuro. Report 3:109-112) and the growth-associated protein B-50/GAP-43 (Skene, 1992, In "The nerve growth cone," Letourneau, Kater and Macagno, Eds.; Coggins and Zwiers, 1991, J. Neurochem. 56:1095-1106).
GAP-43 was originally discovered in an attempt to identify proteins specifically transported into growing axons (Skene and Willard, 1981, J. Cell Biol. 89:96-103; Skene and Willard, 1981, J. Neurosci. 1:419-426; Benowitz et al., 1981, J. Neurosci. 1:300-307). Earlier, a substrate of protein kinase C was isolated which was specific to nervous tissue and this protein was called B-50 (Zwiers et al., 1978, Neurochem. Res. 1:669-677; Zwiers et al., 1980, J. Neurochem. 34:1689-1699; Kristansson et al., 1982, J. Neurochem. 39:371-378). Subsequently, molecular cloning revealed that GAP-43 (Basi et al., 1987, Cell 49:785-791) and B-50 (Nielander et al., 1987, Neurosci. Res. Comm. 1:163-172) are the same protein and are also homologous to F1 (Rosenthal et al., 1987, EMBO J. 6:3611-6346), a calmodulin-binding protein (Cimler et al. 1987, J. Biol. Chem. 262:12158-12263) protein associated with long term potentiation and neuromodulin. An intense interest developed in determining what role, if any, B-50/GAP-43 plays in the development and regeneration of nerve fibers.
Studies on the localization and the regulation of B-50/GAP-43 protein expression and transport have yielded some insights in the role of this protein in axonal growth. The abundance of B-50/GAP-43 in growth cones (Katz et al., 1985, J. Neurosci. 5:1402-1411; De Graan et al., 1985, Neurosci. Lett. 61:235-241; Skene et al., 1986, Science 233:783-786) and developing embryonic neurons (Biffo et al., 1990), coupled with a down regulation of the expression of the protein shortly following target cell innervation, has furthered speculation that B-50/GAP-43 actively participates in the regulation of nerve fiber outgrowth (reviewed in Skene, 1992, supra). B-50/GAP-43 levels in injured PNS neurons are normally upregulated following a lesion and increased levels of B-50/GAP-43 are associated with periods of nerve regeneration (Skene and Willard 1981 (both citations), supra; Verhaagen et al., 1986, Brain Res. Bull. 17:737-741; Hoffman, 1989, J. Neurosci. 9:893-897; Van der Zee et al., 1989, J. Neurosci. 9:3505-3512). The decline in B-50/GAP-43 closely correlates with the completion of synapse formation and with the maturation of axon-glia interactions. This suggest that inhibitory signals associated with these events may play a role in down-regulating B-50/GAP-43 expression. Injury may interrupt this inhibitory influence, resulting in reinduction of B-50/GAP-43 expression (Skene, 1992, supra).
Despite the close correlation between B-50/GAP-43 expression and axonal growth, the precise role of this protein remains elusive. B-50/GAP-43 expression in cultured hippocampal neurons precedes the determination of neuronal polarity (Van Lookeren-Campagne et al., 1992). During the initial stages of nerve fiber elongation, B-50/GAP-43 is equally distributed in all short processes and their growth cones. As polarity in these cultured neurons develops, B-50/GAP-43 becomes more abundant in the faster growing axonal process (Goslin et al., 1988, Nature 336:672-674; Van Lookeren-Campagne et al., 1992, supra). In PC-12 cells treatment with nerve growth factor (NGF) results in a redistribution of B-50/GAP-43 from vesicular structures in the cytosol to the plasma membrane (Van Hooff et al., 1989, J. Cell Biol. 108:1115-1125). This redistribution occurs coincident with the initiation of nerve fiber outgrowth.
Some of the most direct, if undramatic, evidence supporting a role for B-50/GAP-43 in the determination of process outgrowth and cell shape has been obtained in experiments in which the levels of B-50/GAP-43 were manipulated in cell lines. Non-neuronal cells that express B-50/GAP-43 exhibit transient cell surface reactions during the first hours following plating of the cells (Zuber et al., 1989, Science 244:1193-1195; Widmer and Caroni, 1993, J. Cell Biol. 120:503-512). Stable transfection of neuroblastoma cell lines resulted in more rapid neurite outgrowth in response to differentiating stimuli (Yankner et al., 1990, Mol. Brain Res. 7:39-44; Morton and Buss, 1993, Eur. J. Neurosci. 4:910-916) and a longer maintenance of formed processes on withdrawal of such signals (Kumagai et al., 1992, J. Neurochem. 59:41-47). Down regulation of B-50 expression with anti-sense B-50 oligonucleotides or blocking of B-50/GAP-43 with anti-B-50/GAP-43 antibodies results in a diminished outgrowth response in neuroblastoma cells (Shea et al., 1991, J. Neurosci. 11:1685-1690; Jap Tjoen San et al., 1992, Biochem. Biophys. Res. Comm. 187:839-846).
However, it is by no means clear that the B-50/GAP-43 protein has a direct or indirect role in nerve growth or regeneration. For example, collateral motoneuron sprouting of uninjured nerves in response to partial denervation is not associated with increases in GAP-43 mRNA (Brown et al., 1992, Soc. Neurosci. Abstracts 18:605). Burry et al. (ibid.) found that neurite outgrowth was independent of GAP-43 expression, and that NGF stimulation of both events proceeds via different pathways. Similarly, Verhaagen et al. (1993, J. Neurosci. Res. 35:162-169), found that although expression of B-50 mRNA was upregulated in about 40% of olfactory bulb mitral cells following lesioning by transection of the lateral olfactory tract (LOT), enhanced B-50 expression is not accompanied by regeneration of the severed LOT. Platinga et al. (1993, Brain Res. 602:69-76) detected expression of mRNA for B-50 in non-neuronal Schwann cells following sciatic nerve crush without the morphological changes in the Schwann cells that are characteristic of nerve sprouting.
The citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.